Detection and concentration determination of 2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy) propanoic acid by lc/ms/ms

ABSTRACT

A method and system for injecting an unconcentrated sample into a receiving LC/MS/MS system that is configured to determine a concentration of GenX within the unconcentrated sample, wherein the LC/MS/MS includes ESI. The unconcentrated sample is subjected to the following ESI conditions: i) a probe gas temperature of approximately 120° C. to approximately 160° C.; ii) a sheath gas heater setting of approximately 150° C. to approximately 275° C.; and iii) a sheath gas flow of approximately 6 L/min to approximately 11 L/min. The concentration of GenX is determined within the unconcentrated sample, wherein the concentration of GenX within the unconcentrated sample has a minimum reporting level of approximately 0.010 μg/L.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 16/225,363, filed Dec. 19, 2018, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

Embodiments of the present invention relate to qualitative andquantitative analysis of analytes in samples and more particularly tothe qualitative and quantitative analysis of2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy) propanoic acidin water.

BACKGROUND

The fluorochemical2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy) propanoic acid(“GenX acid” or simply “GenX”) is employed in a process (i.e., the “GenXprocess”) that has been used in products such as food packaging, paints,cleaning products, non-stick coatings, outdoor fabrics and firefightingfoam. The GenX process was developed to replace processes that producedother per- and polyfluoroalkyl substances (PFAS) such asperfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS). MostUS industries have phased out production of PFOA and PFOS because ofconcerns about health risks to humans and, instead, have employedprocesses that employ alternative PFAS, such as GenX. Although there isa substantial body of knowledge regarding health risks from older PFASlike PFOS and PFOA, there is much less knowledge about the health risksassociated with new PFAS like GenX.

Recently, GenX has been detected in Cape Fear River near Wilmington,N.C., presumably originating from a plant employing the GenX processupstream from Wilmington. Because of concerns regarding the yet unknownhealth risks to humans exposed to GenX, this event has triggeredsignificant interest in finding inexpensive and sensitive methods fordetecting GenX in other public water sources that are near plants thatemploy the GenX process.

SUMMARY OF INVENTION

Shortcomings of the prior art are overcome and additional advantages areprovided through the provision of a method and system for detecting GenXin a solution or an unconcentrated sample.

The method includes injecting an unconcentrated sample into a receivingLC/MS/MS (liquid chromatography/tandem mass spectroscopy) system, whichis configured to determine a concentration of GenX within theunconcentrated sample, wherein the LC/MS/MS includes electrosprayionization (ESI); subjecting the unconcentrated sample to the followingESI conditions: i) a probe gas temperature of approximately 120° C. toapproximately 160° C., ii) a sheath gas heater setting of approximately150° C. to approximately 275° C., and iii) a sheath gas flow ofapproximately 6 L/min to approximately 11 L/min; and determining theconcentration of GenX within the unconcentrated sample, wherein theconcentration of GenX within the unconcentrated sample is at leastapproximately 0.010 μg/L.

The system includes an LC/MS/MS system operable utilizing ESI andconfigured to receive an injection of an unconcentrated samplecontaining GenX. The LC/MS/MS system subjects the unconcentrated sampleto the following ESI conditions: i) a probe gas temperature ofapproximately 120° C. to approximately 160° C.; ii) a sheath gas heatersetting of approximately 150° C. to approximately 275° C.; and iii) asheath gas flow of approximately 6 L/min to approximately 11 L/min. TheLC/MS/MS system determines a concentration of GenX within theunconcentrated sample, wherein the concentration of GenX within theunconcentrated sample is at least approximately 0.010 μg/L.

In an embodiment, the concentration of GenX within the unconcentratedsample is between approximately 0.010 μg/L to approximately 1.0 μg/L. Inan embodiment, the unconcentrated sample is subjected to the followingESI conditions: i) a probe gas temperature of approximately 120° C.; ii)a sheath gas heater setting of approximately 150° C.; and iii) a sheathgas flow of approximately 6 L/min. In this embodiment, the ESIconditions may also include: i) a negative ion polarity setting; ii) agas flow setting of approximately 11 L/min; iii) a nebulizer setting of20 psi; iv) a capillary voltage setting of approximately 3000 V; v) avoltage charging setting of 0; and vi) the following ion-funnelparameters: high pressure RF=90 and low pressure RF=60. When theseconditions are configured, the method/system may detect GenX within asecond unconcentrated sample, wherein a second concentration of GenXwithin the second unconcentrated sample is at least approximately 0.0022μg/L. In an embodiment, an amount of GenX in a single injection ofunconcentrated sample is at least approximately 7.5×10⁻⁷ μg. In anembodiment, an amount of GenX in a single injection of unconcentratedsample is between approximately 7.5×10⁻⁷ μg to approximately 7.5×10⁻⁵μg.

The method further includes injecting a solution containing GenX into anLC/MS/MS system that is configured to detect GenX within the solution,wherein the LC/MS/MS includes ESI; subjecting a GenX-containing LCeluent of the solution to the following ESI conditions: i) a probe gastemperature of approximately 120° C. to approximately 160° C., ii) asheath gas heater setting of approximately 150° C. to approximately 275°C., and iii) a sheath gas flow of approximately 6 L/min to approximately11 L/min; and determining a concentration of GenX within the solutioncontaining GenX, wherein an injected amount of GenX within the solutioncontaining GenX is at least approximately 7.5×10⁻⁷ μg.

The system further includes an LC/MS/MS system operable utilizing ESIand configured to receive an injection of a solution containing GenX.The LC/MS/MS system subjects a GenX-containing LC eluent of the solutionto the following ESI conditions: i) a probe gas temperature ofapproximately 120° C. to approximately 160° C.; ii) a sheath gas heatersetting of approximately 150° C. to approximately 275° C.; and iii) asheath gas flow of approximately 6 L/min to approximately 11 L/min. TheLC/MS/MS system determines a concentration of GenX within the solutioncontaining GenX, wherein a received injected amount of GenX within thesolution containing GenX is at least approximately 7.5×10⁻⁷ μg.

In an embodiment, the injected amount of GenX within the solutioncontaining GenX is between approximately 7.5×10⁻⁷ μg to approximately7.5×10⁻⁵ μg. In an embodiment, the GenX-containing eluent is subjectedto the following ESI conditions: i) a probe gas temperature ofapproximately 120° C.; ii) a sheath gas heater setting of approximately150° C.; and iii) a sheath gas flow of approximately 6 L/min. In thisembodiment, the ESI conditions may also include: i) a negative ionpolarity setting; ii) a gas flow setting of approximately 11 L/min; iii)a nebulizer setting of 20 psi; iv) a capillary voltage setting ofapproximately 3000 V; v) a voltage charging setting of 0; and vi) thefollowing ion-funnel parameters: high pressure RF=90 and low pressureRF=60. When these conditions are configured, the method/system maydetect GenX within a second injected solution containing GenX, wherein asecond amount of GenX within the second injected solution is at leastapproximately 1.7×10⁻⁷ μg. In an embodiment, the GenX concentration isdetermined to be at least approximately 0.010 μg/L when approximately 75μL of the solution containing GenX is injected into the LC/MS/MS system.In an embodiment, GenX is detected at a concentration of at leastapproximately 0.022 μg/L when approximately 75 μL of the second solutioncontaining GenX is injected into the LC/MS/MS system.

In an embodiment, the unconcentrated sample and solution are aqueousunconcentrated samples and solutions, respectively. In this embodiment,the aqueous unconcentrated sample and solution are selected from:finished drinking water, ground water, raw source water, and water at anintermediate stage of treatment between raw source water and finisheddrinking water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates processes for validating a method to determine levelsof GenX in samples, in accordance with an exemplary embodiment of thepresent invention.

FIG. 2 depicts a block diagram of components of an LC/MS/MS system usedto determine levels of GenX in samples, in accordance with an exemplaryembodiment of the present invention.

DETAILED DESCRIPTION

Currently, the quantitative determination of low levels of the analyteGenX in water sources requires extraction of the GenX from the water dueto the limitations of the analytical methods employed for sampleanalysis. When analyte concentrations are too low to be quantitated,extraction thereof serves to provide a more concentrated sample than thecollected unconcentrated water sample. These extraction steps are oftentime-consuming, costly, and inherently introduce the possibility oferrors in the analysis. In some cases, up to one liter of water from acontaminated water source must be extracted to provide 1 mL of anaqueous sample after evaporation of extracting solvent and subsequentaqueous dissolution of the isolated extract. Furthermore, detection ofGenX by liquid chromatography/tandem mass spectroscopy (LC/MS/MS) isimpeded by the instability of GenX during ionization compared to otherPFAS. This instability results in the breakdown of the compound, whichmakes it difficult to easily detect GenX and quantitate itsconcentration.

Embodiments of the present invention recognize that extraction stepscontribute to increased costs and errors in the qualitative andquantitative analysis of GenX in water samples. Embodiments of thepresent invention recognize that typical ionization conditions, whichprovide quantitative analysis of PFAS such as PFOA and PFOS, lead tocomplex fragmentation of GenX samples; this makes the detection andconcentration determination of GenX in aqueous samples difficult, if notimpossible. Embodiments of the present invention provide a method andLC/MS/MS system for the detection and concentration determination of lowlevels of GenX in unconcentrated as well as concentrated samples. In thecase of unconcentrated samples, such as finished drinking water, groundwater, raw source water, and water at an intermediate stage of treatmentbetween raw source water and finished drinking water, extractiontechniques are avoided. Embodiments of the present invention provideelectrospray ionization (ESI) conditions that avoid complexfragmentation of injected GenX unconcentrated and concentrated samples,thereby making said GenX samples readily subject to low-level GenXdetection and concentration determination.

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function, anduse of the methods and systems disclosed herein. One or more examples ofthese embodiments are illustrated in the accompanying drawings. Thoseskilled in the art will understand that the methods and systemsspecifically described herein and illustrated in the accompanyingdrawings are non-limiting exemplary embodiments and that the scope ofthe present invention is defined solely by the claims. The featuresillustrated or described in connection with one exemplary embodiment maybe combined with the features of other embodiments. Such modificationsand variations are intended to be included within the scope of thepresent invention.

The terms “substantially”, “approximately”, “about”, “relatively,” orother such similar terms that may be used throughout this disclosure,including the claims, are used to describe and account for smallfluctuations, such as due to variations in processing. For example, theycan refer to less than or equal to +10%, such as less than or equal to+5%, such as less than or equal to +2%, such as less than or equal to+1%, such as less than or equal to +0.5%, such as less than or equal to+0.2%, such as less than or equal to +0.1%, such as less than or equalto +0.05%.

In various embodiments, unconcentrated samples are analyzed fordetection and quantitation of the analyte GenX. As used herein,“unconcentrated sample” typically refers to an aqueous sample collectedfrom a water source such as, but not limited to, finished drinkingwater, ground water, raw source water, and water at an intermediatestage of treatment between raw source water and finished drinking water.The sample may also be collected from an effluent from processes thatutilize GenX, such as from a factory that produces GenX-containingproducts. The unconcentrated sample is not concentrated by anydeliberate or substantial evaporation of the solvent, i.e., water.Further, the unconcentrated sample is not concentrated by, for example,extraction into an organic solvent to subsequently make a non-aqueous oraqueous solution of GenX that has higher concentration than theoriginally collected sample. An unconcentrated sample also includes asample that is diluted with respect to the originally collected sample.The diluent may be water or a water-miscible solvent such as, but notlimited to, an alcohol (e.g., methanol, ethanol, n-propanol,isopropanol, n-butanol sec-butanol, iso-butyl alcohol, tert-butylalcohol, diols such as ethylene glycol, triols such as glycerol, etc.),acetonitrile, dimethylformamide, dimethyl sulfoxide, dimethylacetamide,etc. In some embodiments, unconcentrated samples also contain addedchemicals, such as ammonium chloride for purposes of dechlorination.

Unconcentrated samples include such water samples which are not dilutedor concentrated such that they may be directly injected into the systemfor analysis.

In various embodiments, concentrated samples are analyzed for detectionand quantitation of GenX at extremely low levels. As used herein,“concentrated samples” include samples obtained via one or more of thefollowing steps: i) the extraction of GenX from a first volume of water(typically an aqueous sample obtained directly from a water source) intosecond volume of a water-immiscible solvent, wherein the second volumeof a water-immiscible solvent is less than, substantially the same, orgreater than the first volume of water; ii) partial or completeevaporation of the water-immiscible solvent to concentrate the GenXcontained therein; and iii) re-dissolving the GenX analyte into a thirdvolume of water with or without the concomitant introduction ofpreservatives and/or dechlorination agents, wherein the third volume ofwater is of a lesser volume than the first volume of water.

In some embodiments of the present invention, concentrated andunconcentrated samples of GenX include samples collected and preparedfrom soil and plants, as described elsewhere for PFAS that do notinclude GenX (e.g., see Huset and Barry, “Quantitative determination ofperfluoroalkyl substances (PFAS) in soil, water, and home gardenproduce”, MethodsX 5 (2018) 697-704). In some embodiments, concentratedand unconcentrated samples of GenX include samples collected from urineand blood.

As used herein, the term “GenX solution,” “GenX in a solution,” “asolution containing GenX,” etc. includes a homogeneous solution of GenX,which includes concentrated and unconcentrated GenX samples as well asstandards, etc. As is well-known in the art, for any analyte to beinjected onto an LC/MS/MS system, it must be in a homogeneous solutionof a solvent suitable for injection onto an LC column.

It is understood that within a known volume of an analyte solution thathas a known concentration, the amount of analyte is also known andreadily calculated. For example, 75 microliters (μL or μl) of a GenXsolution that has a concentration of 0.010 micrograms per liter (μg/L orμg/μl) contains 7.5×10⁻⁷ μg of GenX according to the equation: (0.010μg/L)×(75 μL)×(10⁻⁶ L/μL)=7.5×10⁻⁷ μg. Thus, 75 μL of a 0.0022 μg/Lsolution of GenX contains 1.7×10⁻⁷ μg of GenX and 75 μL of a 1.0 μg/Lsolution of GenX contains 7.5×10⁻⁵ μg of GenX. Herein, any known volumeof an analyte solution with a known concentration may be expressed interms of a known mass of analyte.

Herein, analyte concentration may be expressed as parts per trillion(ppt) according to the relationship 1 ng/L=1 ppt. Thus, 0.010 μg/L maybe expressed as 10 ppt, 0.0022 μg/L may be expressed as 2.2 ppt, and 1.0μg/L may be expressed as 1000 ppt. Because the relationship between pptand μg/L is known, a known volume containing a known ppt of an analytemay also be expressed in terms of a known mass of the analyte.

Embodiments of the present invention will now be described in detailwith reference to FIG. 1.

FIG. 1 illustrates processes, generally designated 100, for validating amethod to determine levels of GenX in samples in accordance with anexemplary embodiment of the present invention.

Table 1 abbreviations are used in various tables referred to throughoutFIG. 1.

TABLE 1 Abbreviations WS Working Standard(s) ICS Initial CalibrationStandard(s) CCV Continuing Calibration Verification standard(s) CCV HLContinuing Calibration Verification standard High Level CCV LLContinuing Calibration Verification standard Low Level CCV ML ContinuingCalibration Verification standard Medium Level LFB Laboratory FortifiedBlank(s) LFBML Laboratory Fortified Blank Medium Level LFM LaboratoryFortified Matrix standard(s) LFM ML Laboratory Fortified Matrix standardMedium Level LFMD Laboratory Fortified Matrix Duplicate standard(s) QCQuality Control QCS Quality Control Sample PDS Primary Dilution StandardSS Stock Standard PRW Preserved Reagent Water MB Method Blank ISInternal Standard IDC Initial Demonstration of Capability DOCDemonstration of Capability MDL Method Detection Limit RDL RequiredDetection Limit MRL Minimum Reporting Level RL Reporting Limit

In step 105, working standards (WS) of the analyte GenX are prepared. Invarious embodiments, working standards are instrument calibration,calibration verification, and quality control standards analyzed in ananalytical run such as an initial calibration standard (ICS), acontinuing calibration verification (CCV) standard, a laboratoryfortified blank (LFB), a laboratory fortified matrix (LFM) standard, alaboratory fortified matrix duplicate (LFMD) standard, a quality controlsample (QCS), etc.

An initial calibration standard includes a solution prepared from theprimary dilution standard solution (PDS) or stock standard (SS)solutions. The initial calibration standard solutions are used tocalibrate an instrument response with respect to an analyteconcentration.

A continuing calibration verification includes a calibration standardcontaining a specified concentration of method analytes, which isanalyzed at specified periods to verify an accuracy of the existingcalibration for said analytes.

A laboratory fortified blank is an aliquot of preserved reagent water(PRW) to which known quantities of the method analytes are added in thelaboratory. In various embodiments, the laboratory fortified blank isanalyzed using the same protocol as a sample. In some embodiments, thepurpose of the laboratory fortified blank is to determine whether themethod is valid, and whether the method can make accurate and precisemeasurements with respect to a specified criterion. For some embodimentsof the present invention, there is no substantially significantdifference between a laboratory fortified blank and a continuingcalibration verification standard.

Preserved reagent water is a solution comprising approximately 200 mg/Lammonium chloride solution in deionized water, which is typicallyprepared monthly. Deionized water is water having a resistance ofapproximately 18.2 Megaohms/cm or greater because of deionization.

A laboratory fortified matrix standard is an aliquot of an environmentalsample to which known quantities of method analytes are added in thelaboratory. The laboratory fortified matrix standard is analyzed like asample, and its purpose is to determine whether the sample matrixcontributes bias to the analytical results. The backgroundconcentrations of the analytes in the sample matrix should preferably bedetermined in a separate aliquot and the measured values in thelaboratory fortified matrix standard corrected for backgroundconcentrations. In various embodiments, a laboratory fortified matrixduplicate standard is a second aliquot of an environmental sample usedto prepare the laboratory fortified matrix standard. The laboratoryfortified matrix duplicate standard is fortified, processed, andanalyzed in the same way as the laboratory fortified matrix standard.The laboratory fortified matrix standard duplicate is used instead of alaboratory duplicate to assess method precision when the occurrence oftarget analytes is low.

A quality control sample is a solution of method analytes obtained froma source external to the laboratory and different from the source ofcalibration standards. The quality control sample is used to verify theaccuracy of the calibration standards.

A primary dilution standard is a solution of one or several analytesprepared in the laboratory from stock standard solutions and diluted asneeded to prepare calibration solutions and other needed analytesolutions. Primary dilution standards are prepared at concentrationsthat are suitable for secondary or working standard preparation. Primarydilution standards are typically stored in the refrigerator at ≤4° C.with expiration dates of 3 months. The standard identification,preparation date, expiration date and analyst initials are written onthe label. The expiration date of the prepared standard typically doesnot exceed the expiration date provided by the vendor in its certificateof analysis.

A stock standard is a concentrated solution containing one or moremethod analytes prepared in the laboratory using assayed referencematerials or purchased as certified from the reputable commercialsource. Certified standards are used to prepare primary dilutionstandards, secondary dilution standards and working standards. Invarious embodiments, if certified standards are not available, the solidcompounds are obtained from the manufacturer. In these embodiments, ifcompounds used to prepare solutions are 96% pure or greater, the solidweight is used without correction for purity to calculate theconcentration of the stock standard.

Method blanks are aliquots of preserved reagent water that are treatedexactly as a sample including exposure to all glassware, equipment,solvents, reagents, etc. method blanks are used with other samples. Invarious embodiments, the method blanks are used to determine if methodanalytes or other interferences are present in the laboratoryenvironment, the reagents, or the apparatus.

An internal standard is a pure compound added to a standard solution ina known amount and used to measure the relative response of the methodanalyte. In some embodiments, the internal standard includesisotopically labeled analogues (e.g., ¹³C) of method analyte.

An analysis batch is a set of up to 20 field samples (not includingquality control samples such as method blanks, continuing calibrationverification standards, laboratory fortified matrix standards andlaboratory fortified matrix duplicate standards) that are analyzed onthe same instrument during a 24-hour period that begins and ends withthe analysis of the appropriate continuing calibration verificationstandard. In some embodiments, an additional continuing calibrationverification standard is required after analysis of 10 field samples.

In general, working standards are obtained from materials purchased fromvendors and any products made with TEFLON® are avoided when the analytesbeing tested for include PFAS such as GenX.

Table 2 shows an example of a preparation of stock standards and primarydilution standards for GenX:

TABLE 2 Example of Preparation of Stock Standards and Primary Dilutionfor GenX. Stock Standard Custom Mix-SS Primary dilution Standards PDSVolume Volume Final and of SS Volume/ Analyte Weight Solvent Conc. usedConc Solvent Mix description Name [g] [mL] μg/ml [μL] [μg/mL] [mL] CodeWellington Labs GenX NA NA 50.0 50.0 0.10 25.00 PDS individual ¹³C-GenXNA NA 50.0 25.0 0.050 25.00 PDS IS compounds Apollo Scientific Neat GenX0.010 20.00 500 5.00 0.10 25.00 PDS 2 MeOH Methanol (MeOH) is of LCMSgrade.

Table 3 shows an example of a preparation of working standards for GenX:

TABLE 3 Example of Preparation of Working Standards for GenX. WS Volumeof Final WS Final PDS/ICS Used Volume Solvent Concentration WS Name [μL][mL] Used [μg/L] ICS 7/CCV HL  100 of PDS 10.00 Preserved 1.00 ICS 1/CCVLL 10.0 of ICS 7 1.00 reagent 0.010 ICS 2 25.0 of ICS 7 1.00 water 0.025ICS 3 50.0 of ICS 7 1.00 0.050 ICS 4  100 of ICS 7 1.00 0.10 ICS 5/CCVML  250 of ICS 7 1.00 0.25 ICS 6  500 of ICS 7 1.00 0.50 MDL 5.00 of ICS7 1.00 0.0050 LFB ML (for DOC)  250 of ICS 7 1.00 0.25 QCS 25.0 of PDS 210.00 0.25 LFM/LFMD 25.0 of PDS 10.00 Sample 0.25 10.0 μL of IS is addedto 1.00 mL of each WS resulting in concentration of 0.50 μg/L.

In this example, initial calibration standard 7 (ICS 7), the qualitycontrol sample (QCS), continuing calibration verification standards (CCVHL, ML and LL), and laboratory fortified matrix standards (LFB ML,LFM/LFMD) are prepared by adding determined volumes of primary dilutionstandard solutions to the stock blank or sample. Initial calibrationstandard 1 (ICS 1) through initial calibration standard 6 (ICS 6) aremade by serial dilution of ICS 7 directly into a 2 mL vial. In variousembodiments, the working standards are made fresh for every run. Initialcalibration standard 7, the continuing calibration verificationstandards, the quality control sample and the method blank are typicallytransferred to 2 ml vials prior to analysis.

In step 110, the exact mass of the compounds (analytes/precursor ions)and their product ions is determined by high resolution massspectroscopy.

In step 115, the settings are determined for gas flow and thetemperature of the source of the mass spectrometer. Table 4 shows anexample of settings determined for electrospray ionization (ESI) on anAGILENT 6495 mass spectrometer in step 115:

TABLE 4 Example of LC/MS/MS ESI Conditions developed for GenX. ESIConditions Polarity Negative ion Gas Temp (° C.) 120 Gas Flow (l/min) 11Nebulizer (psi) 20 Sheath Gas Heater 150 Sheath Gas Flow 6 Capillary (V)3000 V Charging 0 Ion Funnel Parameters High Pressure RF 90 Low PressureRF 60

ESI is a technique used in mass spectrometry to produce ions using anelectrospray in which a high voltage is applied to a liquid to create anaerosol that is ionized.

In Table 4, “Polarity” refers to an applied electrical field duringionization, which causes either positive or negative ions to beproduced. “Gas Temp (° C.)” refers to a temperature in Celsius of aninert drying gas (typically nitrogen) that is used to promote theremoval of solvent from aerosol particles in spray ionization. “Gas Flow(1/min)” refers to the volume per unit time (e.g. liters/minute orL/min) that the drying gas is dispersed. “Nebulizer (psi)” refers to thepressure (psi) utilized for the mass spectrometer nebulizer, whichdelivers a fine mist using the specified pressure. “Sheath Gas Heater”refers a temperature setting (° C.) for heating a sheath gas, which isan inert gas (typically nitrogen) introduced through a tube that iscoaxial with the electrospray emitter to pneumatically assist theformation of the sprayed droplets. “Sheath Gas Flow” refers to thevolume per unit time (e.g. liters/minute or L/min) that the sheath gasis dispersed. “Capillary (V)” refers to a voltage (in volts) applied tothe tip of a metal capillary relative to the surrounding source-samplingcone or heated capillary. This strong electric field causes thedispersion of the sample solution into an aerosol of highly chargedelectrospray droplets. “V charging” refers to a setting for a chargingelectrode in the instrument, in this example the instrument is anAGILENT 6495 mass spectrometer. “Ion Funnel Parameters” refers tosettings for an ion funnel, which is used to focus a beam of ions usinga series of stacked ring electrodes with decreasing inner diameter. Acombined radio frequency (RF) and fixed electrical potential is appliedto the grids.

In various embodiments, ESI conditions include a gas temperature setting(“Gas Temp (° C.)”) of approximately 120° C. to approximately 160° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 120° C. to approximately 155° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 120° C. toapproximately 150° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 120° C. to approximately 145° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 120° C. to approximately 140° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 120° C. toapproximately 135° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 120° C. to approximately 130° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 120° C. to approximately 125° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 125° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 125° C. toapproximately 155° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 125° C. to approximately 150° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 125° C. to approximately 145° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 125° C. toapproximately 140° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 125° C. to approximately 135° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 125° C. to approximately 130° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 125° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 130° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 130° C. toapproximately 155° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 130° C. to approximately 150° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 130° C. to approximately 145° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 130° C. toapproximately 140° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 130° C. to approximately 135° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 130° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 135° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 135° C. toapproximately 155° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 135° C. to approximately 150° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 135° C. to approximately 145° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 135° C. toapproximately 140° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 135° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 140° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 140° C. toapproximately 155° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 140° C. to approximately 150° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 140° C. to approximately 145° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 140° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 145° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 145° C. toapproximately 155° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 145° C. to approximately 150° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 145° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 150° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 150° C. toapproximately 155° C. In some embodiments, ESI conditions include a gastemperature setting of approximately 150° C.

In some embodiments, ESI conditions include a gas temperature setting ofapproximately 155° C. to approximately 160° C. In some embodiments, ESIconditions include a gas temperature setting of approximately 155° C. Insome embodiments, ESI conditions include a gas temperature setting ofapproximately 160° C.

In exemplary embodiments, ESI conditions include a gas temperaturesetting of approximately 120° C.

In some embodiments, the ESI gas temperature setting is set on anAGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, theESI gas temperature setting is set on an AGILENT 6495 mass spectrometer.

In various embodiments, ESI conditions include a sheath gas heatersetting (“Sheath Gas Heater”) of approximately 150° C. to approximately275° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C. to approximately 270° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 150° C. to approximately 265° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 150° C.to approximately 260° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 150° C. to approximately 255°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C. to approximately 250° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 150° C. to approximately 245° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 150° C.to approximately 240° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 150° C. to approximately 235°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C. to approximately 230° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 150° C. to approximately 225° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 150° C.to approximately 220° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 150° C. to approximately 215°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C. to approximately 210° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 150° C. to approximately 205° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 150° C.to approximately 200° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 150° C. to approximately 195°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C. to approximately 190° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 150° C. to approximately 185° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 150° C.to approximately 180° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 150° C. to approximately 175°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C. to approximately 170° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 150° C. to approximately 165° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 150° C.to approximately 160° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 150° C. to approximately 155°C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 155° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 155°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 155° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 155° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 155° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 155° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 155° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 155° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 155° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 155° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 155° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 155° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 155° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 155° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 155° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 155° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 155° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 155° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 155° C. to approximately 185°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 155° C. to approximately 180° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 155° C. to approximately 175° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 155° C.to approximately 170° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 155° C. to approximately 165°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 155° C. to approximately 160° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 155° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 160° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 160°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 160° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 160° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 160° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 160° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 160° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 160° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 160° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 160° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 160° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 160° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 160° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 160° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 160° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 160° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 160° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 160° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 160° C. to approximately 185°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 160° C. to approximately 180° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 160° C. to approximately 175° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 160° C.to approximately 170° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 160° C. to approximately 165°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 160° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 165° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 165°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 165° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 165° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 165° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 165° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 165° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 165° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 165° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 165° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 165° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 165° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 165° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 165° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 165° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 165° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 165° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 165° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 165° C. to approximately 185°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 165° C. to approximately 180° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 165° C. to approximately 175° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 165° C.to approximately 170° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 165° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 170° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 170°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 170° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 170° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 170° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 170° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 170° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 170° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 170° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 170° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 170° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 170° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 170° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 170° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 170° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 170° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 170° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 170° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 170° C. to approximately 185°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 170° C. to approximately 180° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 170° C. to approximately 175° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 170° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 175° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 175°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 175° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 175° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 175° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 175° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 175° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 175° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 175° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 175° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 175° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 175° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 175° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 175° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 175° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 175° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 175° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 175° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 175° C. to approximately 185°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 175° C. to approximately 180° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 175° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 180° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 180°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 180° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 180° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 180° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 180° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 180° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 180° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 180° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 180° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 180° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 180° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 180° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 180° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 180° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 180° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 180° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 180° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 180° C. to approximately 185°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 180° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 185° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 185°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 185° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 185° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 185° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 185° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 185° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 185° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 185° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 185° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 185° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 185° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 185° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 185° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 185° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 185° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 185° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 185° C.to approximately 190° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 185° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 190° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 190°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 190° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 190° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 190° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 190° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 190° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 190° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 190° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 190° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 190° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 190° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 190° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 190° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 190° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 190° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 190° C. to approximately 195° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 190° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 195° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 195°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 195° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 195° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 195° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 195° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 195° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 195° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 195° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 195° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 195° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 195° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 195° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 195° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 195° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 195° C. to approximately 200° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 195° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 200° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 200°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 200° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 200° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 200° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 200° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 200° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 200° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 200° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 200° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 200° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 200° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 200° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 200° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 200° C. to approximately 205°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 200° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 205° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 205°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 205° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 205° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 205° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 205° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 205° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 205° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 205° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 205° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 205° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 205° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 205° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 205° C.to approximately 210° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 205° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 210° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 210°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 210° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 210° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 210° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 210° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 210° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 210° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 210° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 210° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 210° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 210° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 210° C. to approximately 215° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 210° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 215° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 215°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 215° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 215° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 215° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 215° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 215° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 215° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 215° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 215° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 215° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 215° C. to approximately 220° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 215° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 220° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 220°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 220° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 220° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 220° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 220° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 220° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 220° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 220° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 220° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 220° C. to approximately 225°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 220° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 225° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 225°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 225° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 225° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 225° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 225° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 225° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 225° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 225° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 225° C.to approximately 230° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 225° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 230° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 230°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 230° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 230° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 230° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 230° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 230° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 230° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 230° C. to approximately 235° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 230° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 235° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 235°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 235° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 235° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 235° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 235° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 235° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 235° C. to approximately 240° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 235° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 240° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 240°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 240° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 240° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 240° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 240° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 240° C. to approximately 245°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 240° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 245° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 245°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 245° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 245° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 245° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 245° C.to approximately 250° C. In some embodiments, ESI conditions include asheath gas heater setting of approximately 245° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 250° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 250°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 250° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 250° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 250° C. to approximately 255° C. In some embodiments, ESIconditions include a sheath gas heater setting of approximately 250° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 255° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 255°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 255° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 255° C. to approximately 260° C. In someembodiments, ESI conditions include a sheath gas heater setting ofapproximately 255° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 260° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 260°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 260° C. to approximately265° C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 260° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 265° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 265°C. to approximately 270° C. In some embodiments, ESI conditions includea sheath gas heater setting of approximately 265° C.

In some embodiments, ESI conditions include a sheath gas heater settingof approximately 270° C. to approximately 275° C. In some embodiments,ESI conditions include a sheath gas heater setting of approximately 270°C. In some embodiments, ESI conditions include a sheath gas heatersetting of approximately 275° C.

In exemplary embodiments, ESI conditions include a sheath gas heatersetting of approximately 150° C.

In some embodiments, the ESI sheath gas heater setting is set on anAGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, theESI sheath gas heater setting is set on an AGILENT 6495 massspectrometer.

In various embodiments, ESI conditions include a sheath gas flow(“Sheath Gas Flow”) of approximately 6 L/min to approximately 11 L/min.In some embodiments, ESI conditions include a sheath gas flow ofapproximately 6 L/min to approximately 10 L/min. In some embodiments,ESI conditions include a sheath gas flow of approximately 6 L/min toapproximately 9 L/min. In some embodiments, ESI conditions include asheath gas flow of approximately 6 L/min to approximately 8 L/min. Insome embodiments, ESI conditions include a sheath gas flow ofapproximately 6 L/min to approximately 7 L/min.

In some embodiments, ESI conditions include a sheath gas flow ofapproximately 7 L/min to approximately 11 L/min. In some embodiments,ESI conditions include a sheath gas flow of approximately 7 L/min toapproximately 10 L/min. In some embodiments, ESI conditions include asheath gas flow of approximately 7 L/min to approximately 9 L/min. Insome embodiments, ESI conditions include a sheath gas flow ofapproximately 7 L/min to approximately 8 L/min. In some embodiments, ESIconditions include a sheath gas flow of approximately 7 L/min.

In some embodiments, ESI conditions include a sheath gas flow ofapproximately 8 L/min to approximately 11 L/min. In some embodiments,ESI conditions include a sheath gas flow of approximately 8 L/min toapproximately 10 L/min. In some embodiments, ESI conditions include asheath gas flow of approximately 8 L/min to approximately 9 L/min. Insome embodiments, ESI conditions include a sheath gas flow ofapproximately 8 L/min.

In some embodiments, ESI conditions include a sheath gas flow ofapproximately 9 L/min to approximately 11 L/min. In some embodiments,ESI conditions include a sheath gas flow of approximately 9 L/min toapproximately 10 L/min. In some embodiments, ESI conditions include asheath gas flow of approximately 9 L/min.

In some embodiments, ESI conditions include a sheath gas flow ofapproximately 10 L/min to approximately 11 L/min. In some embodiments,ESI conditions include a sheath gas flow of approximately 10 L/min. Insome embodiments, ESI conditions include a sheath gas flow ofapproximately 11 L/min.

In exemplary embodiments, ESI conditions include a sheath gas flow ofapproximately 6 L/min.

In some embodiments, the ESI sheath gas flow setting is set on anAGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, theESI sheath gas flow setting is set on an AGILENT 6495 mass spectrometer.

In step 120, aqueous standards as described supra are prepared for thedetermination of chromatographic conditions.

In step 125, a chromatographic gradient is developed for the compounds(analytes). In one embodiment of a developed chromatographic gradient,the column used is an analytical column: ZORBAX ECLIPSE PLUS C18, 2.1×50mm, 1.8 um. The following conditions with the column were used: Columntemperature=40° C.; injection volume=75 μL; and flow=0.30 mL/min. Table5 shows an example of a developed GenX gradient method on this columnunder these parameters:

TABLE 5 Example of an LC Gradient for GenX. LC Gradient Program-NegativeIons % 5 mM ammonium Time (min) acetate % Methanol 0 95 5 1 5 95 2 5 952.2 95 5 3.2 95 5

In step 130 the chromatographic gradient developed in step 125 iscombined with the mass spectroscopy settings determined in step 115.Table 6 shows an example of triple quadrapole MS/MS method conditionsfor GenX after combination with the Table 5 LC gradient:

TABLE 6 Example of LC/MS/MS Method Conditions for GenX. TripleQuadrupole MS/MS Method Conditions Retention Precursor Product CollisionCell Scan Time Ion Ion MS1 Frag Energy Acceleration Analyte Type (min)(m/z) (m/z)^(a) MS2 Voltage (ev)^(b) (V) GenX Primary 2.44 329 168.9Unit 380 5 1 GenX Qualifier 2.44 329 285 Unit 380 1 1 GenX-C13 Primary2.44 332 287 Unit 380 1 1 ^(a)Ions used for quantitation purposes.^(b)Nitrogen used as collision gas

For Table 6, the precursor ion is the deprotonated molecule ([M-H]⁻) ofthe target analyte. In MS/MS, the precursor ion is mass selected andfragmented by collisionally activated dissociation to producedistinctive product ions of smaller m/z. The product ion is one of thefragment ions produced in MS/MS by the collisionally activateddissociation of the precursor ion. In this example, the GenX primary ionis 168.9 and the GenX qualifier ion is 285. However, either ion can beused for primary and qualifier ions, i.e., the GenX primary ion may be285 and the GenX qualifier ion may be 168.9.

In the examples shown in Tables 4-6 the following instrumentation isused:

i) AGILENT LC/MS/MS System (Column: Analytical column ZORBAX

ECLIPSE PLUS C18, 2.1×50 mm, 1.8 um);

ii) AGILENT 1290 INFINITY Autosampler;

iii) AGILENT 1290 Binary Pump;

iv) AGILENT 1290 TCC Column Compartment; and

v) AGILENT 6495 Mass Spectrometer.

In the examples shown in Tables 4-6 the data software used is AGILENTMASS HUNTER.

In steps 135 and 140, a practical range of detection is determined usingcalibration standards prepared as described supra and method validationsamples. In these steps, quality control (QC) includes a demonstrationof capability (DOC) requirement and a determination of the methoddetection limit (MDL). Ongoing QC requirements are continuously met whenpreparing and analyzing samples.

In various embodiments, an initial demonstration of capability (IDC) isperformed prior to analyzing any field samples and any time major methodmodifications are made. The following steps are exemplary:

i) Generate an acceptable instrument calibration and demonstrate a lowsystem background by analyzing an acceptable method blank. The massspectrometer is calibrated according to the manufacturer'srecommendations. Prior to the analysis of samples, the instrument'sperformance is optimized, and an instrument calibration curve isgenerated. The instrument is calibrated using standards at seven (i.e.,ICS1-ICS7) concentrations, as listed in Table 3. They are analyzed withevery analytical run. ICS1 contains the analyte at a concentration equalto or below the minimum reporting level (MRL). A calibration curve isgenerated for each analyte by plotting the responses against knownconcentrations. In preferred embodiments, linear and quadraticregression models are used. Both weighted and unweighted models areused. In various embodiments, a calibration curve regression model and arange of calibration levels is used for all routine sample analysis. Theinitial calibration is verified by analyzing various concentrations ofCCV ((low level) LL, (medium level) ML, (high level) HL) prior to sampleanalysis and after every 10 samples (see Table 11 below).ii) Analyze a method blank to demonstrate low background contamination(an example of method blank (MB) acceptance criteria is shown in Table11 below).iii) Demonstrate method precision and accuracy by analyzing 4 replicatesof a laboratory fortified method blank medium level (LFBML) prepared asindicated in Table 3 and as described supra. The acceptance criteria areas follows: relative percent difference (RPD) <20% and accuracy as meanpercent recovery is within +30%.iv) Establish the method detection limit (MDL) by analyzing sevenreplicates of laboratory fortified blank (LFB) fortified at less than orequal to the concentration of the reporting limits (RL) listed in Table10. In some embodiments, the MDL study is performed over a minimum of 3days. The determination of the MDL is described in more detail infra.v) The MDL verification is performed at the time of initial methoddevelopment, each time the MDL study is performed, and on an annualbasis. The LFB at a concentration of 2-3 times the calculated MDL value(but less than RL and concentration used for MDL study and less than orequal to the required detection levels (RDL) if applicable) is preparedand analyzed. The analyte must be positively identified. If an analyteidentification cannot be confirmed at the prepared concentration theinstrument shall be maintained to restore sensitivity or the RL of thisanalyte must be re-evaluated.

Ongoing quality control applied when performing this method includesanalyzing acceptable instrument calibration/calibration verificationstandards, MB, QCS, LFM, and LFMD with tested samples at the frequencyand acceptance criteria required.

Table 11 (infra) illustrates an example of a laboratory analytical runsequence for this method, with QC parameters frequency, concentrationsand acceptance criteria.

An example of a DOC Study including the demonstration of laboratoryprecision and accuracy are presented in Table 7 using 75 μL injectionvolumes:

TABLE 7 Example of a DOC Study for GenX. Data File 17 18 19 20 AccuracyMethod's Method's Amount Amount Recovered as Mean Accuracy StandardPrecision Precision Analyte Added LFB ** LFB** LFB** LFB** Mean RecoveryLimits Deviation as RSD* Limits Name [μg/L] [μg/L] [μg/L] [μg/L] [μg/L][μg/L] [%] [%] [μg/L] [%] [%] GenX 0.250 0.2532 0.2532 0.2571 0.25360.254 101.7 70.0-130.0 0.0019 0.7 <20.0

Determination of MDL

MDL (Method Detection Limits) are the minimum concentration of asubstance that can be reported with 99% confidence that the measuredconcentration is distinguishable from Method Blank results. An exampleof a procedure for determining MDL is as follows:

First, an estimate is made of an initial MDL using one or more of: i) amean determined concentration plus three times the standard deviation ofa set of MB; ii) a concentration value that corresponds to an instrumentsignal/noise in the range of 3 to 5; iii) a concentration equivalent ofthree times the standard deviation of replicate instrumentalmeasurements of spiked blanks; iv) a region of the calibration wherethere is a significant change in sensitivity, such as a break in theslope of the calibration; v) an instrumental limitation; and vi) apreviously determined MDL.

Second, an initial MDL determination is made by selecting a spikinglevel, typically 2-10 times the estimated method detection limit fromabove, but less than the value of the laboratory established RL and lessthan or equal to a regulatory authority reported required detectionlimit (RDL), if one exists. Once the spiking level is determined, aminimum of seven laboratory standards in reagent water (containing allmethod preservatives, if applicable) are made at the selected spikinglevel concentration and they are processed through all steps of themethod. Generally, the standards used for the MDL are prepared in atleast three batches on three separate calendar dates and analyzed onthree separate calendar dates. Preparation and analysis may be performedon the same day. In general, statistical outlier removal procedures arenot used to remove data for the initial MDL determination since thetotal number of observations is small and the purpose of the MDLprocedure is to capture routine method variability. However, documentedinstances of gross failures (e.g., instrument malfunctions, mislabeledsamples, cracked vials) may be excluded from the calculations, providedthat at least seven spiked samples and seven method blanks areavailable. After the method is run, the spiking level is evaluated. Ifany result for any individual analyte from the spiked samples does notmeet a qualitative method identification criterion or does not provide anumerical result greater than zero, then the method is repeated withspiked samples at a higher concentration.

The method MDL is the greater of either an MDL based on spiked samples(MDLs) or an MDL based on method blanks (MDLb).

The MDLs is calculated as shown below:

First, a mean of the measured concentration values X is calculated asshown below:

$X = {\Sigma \frac{Xi}{n}}$

Where:

-   -   i=from 1 to n;    -   n=the number of data points; and Xi=the measured concentration        value of an individual laboratory standard.

Second, a mean percent recovery (R) is calculated as shown below:

$R = {\frac{X}{T} \times 100\%}$

Where:

-   -   X=mean of the measured concentration values; and    -   T=true concentration used.

Third, a standard deviation (Ss) is calculated as shown below:

${Ss} = \left. \sqrt{}\frac{{\Sigma \left( {{Xi} - X} \right)}^{2}}{n - 1} \right.$

Where:

-   -   i=from 1 to n    -   n=the number of data points;    -   Xi=the measured concentration value of an individual laboratory        standard; and    -   X=mean of the measured concentration values.

The MDLs is then calculated as shown below:

MDLs=t _((n-1, 1-α⇄0.99)) *Ss

Where:

-   -   t_((n−1, 1−α=0.99))=the Student's t-value appropriate for a        single-tailed 99^(th) percentile t statistic and a standard        deviation estimate within −1 degrees of freedom (see Table 8        below); and    -   Ss=standard deviation of the replicate spiked sample analyses.

For the MLDb, one of the following criterion is applied:

i) If none of the method blanks give numerical results for an individualanalyte, the MDLb does not apply and the MDLs is used. A numericalresult includes both positive and negative results, including resultsbelow a current MDL, but not results of “ND” (not detected) commonlyobserved when a peak is not present in chromatographic analysis;ii) If some (but not all) of the method blanks for an individual analytegive numerical results, set the MDLb equal to the highest method blankresult; oriii) If all of the method blanks for an individual analyte givenumerical results, then the MDLb is calculated as shown below:First, a mean of the measured concentration values X is calculated asshown below:

$X = {\Sigma \frac{Xi}{n}}$

Where:

-   -   i=from 1 to n;    -   n=the number of data points; and    -   Xi=the measured concentration value of an individual MB.

Second, a standard deviation (Sb) is calculated as shown below:

${Sb} = \left. \sqrt{}\frac{{\Sigma \left( {{Xi} - X} \right)}^{2}}{n - 1} \right.$

Where:

-   -   i=from 1 to n    -   n=the number of data points;    -   Xi=the measured concentration value of an individual MB; and    -   X=mean of the measured MB concentration values.

Third, the MDLb is then calculated as shown below:

MDLb=X+t _((n−1,1−α=0.99)) *Sb

Where:

-   -   X=mean of the MB results (zero is used in place of the mean if        the mean is negative);    -   t_((n−1, 1−α=0.99))=the Student's t-value appropriate for a        single-tailed 99^(th) percentile t statistic and a standard        deviation estimate within −1 degrees of freedom (see Table 8        below); and    -   Sb=standard deviation of the MB analyses.

TABLE 8 Student's Single-Tailed 99^(th) Percentile t Statistic Values.Degrees Replicate of Student's Number Freedom t-Value n n-1t_((n-1, 0.99)) 7 6 3.143 8 7 2.998 9 8 2.896 10 9 2.821 11 10 2.764 1211 2.718 13 12 2.681 14 13 2.650 15 14 2.624 16 15 2.602 17 16 2.583 1817 2.567 19 18 2.552 20 19 2.539 21 20 2.528 22 21 2.518 23 22 2.508 2423 2.500 25 24 2.492 26 25 2.485 27 26 2.479 28 27 2.473 29 28 2.467 3029 2.462 31 30 2.457 32 31 2.453 33 32 2.449 34 33 2.445 35 34 2.441 3635 2.438 37 36 2.434 38 37 2.431 39 38 2.429 40 39 2.426 41 40 2.423 4241 2.421 43 42 2.418 44 43 2.416 45 44 2.414 46 45 2.412 47 46 2.410 4847 2.408 49 48 2.407 50 49 2.405 51 50 2.403 52 51 2.402 53 52 2.400 5453 2.399 55 54 2.397 56 55 2.396 57 56 2.395 58 57 2.394 59 58 2.392 6059 2.391 61 60 2.390 62 61 2.389 63 62 2.388 64 63 2.387 65 64 2.386 6665 2.385 67 66 2.384 68 67 2.383 69 68 2.382 70 69 2.382 71 70 2.381 7271 2.380 73 72 2.379 74 73 2.379 75 74 2.378 76 75 2.377 77 76 2.376 7877 2.376 79 78 2.375 80 79 2.374 81 80 2.374 82 81 2.373 83 82 2.373 8483 2.372 85 84 2.372 86 85 2.371 87 86 2.370 88 87 2.370 89 88 2.369 9089 2.369 91 90 2.368 92 91 2.368 93 92 2.368 94 93 2.367 95 94 2.367 9695 2.366 97 96 2.366 98 97 2.365 99 98 2.365 100 99 2.365 101 100 2.364∞ ∞ 2.326

An example of an MDL study for GenX is shown in Table 9 using 75 μLinjection volumes.

TABLE 9 Example of a GenX MDL study. Preparation/ Jan. 30, 2018 14 15 16NA NA NA NA Analysis Data #1 & Run/Data Files: Preparations/ Jan. 31,2018 NA NA NA 14 15 NA NA Analysis Data #2 & Run/Data Files:Preparations/ Feb. 1. 2018 NA NA NA NA NA 34 15 Analysis Data #3 &Run/Data Files: Std. Spiked Observed Concentration Mean Dev. Conc. MDLMDL MDL MDL MDL MDL MDL Conc. R (Sa) MDLa RL Analysis [μg/L] [μg/L][μg/L] [μg/L] [μg/L] [μg/L] [μg/L] [μg/L] [μg/L] [%] [μg/L] [μg/L][μg/L] GenX 0.050 0.00480 0.00527 0.00427 0.00621 0.00603 0.005740.00558 0.0054 108.3 0.00069 0.0022 0.010 Data points count: 1 2 3 4 5 67

In general, an MDL verification is performed each time an MDL study isperformed and on an annual basis. In one scenario, if an MDL value isgreater than or equal to the concentration used for the MDL study, theconcentration used for MDL study will be the MDL verificationconcentration. In another scenario, if an MDL value is less than theconcentration used for MDL study, a laboratory standard is prepared andanalyzed in reagent water (with preservatives if applicable), whereinthe, laboratory standard prepared has an analyte concentration:

i) greater than or equal to the MDL value;

ii) no more than 2-3 times the MDL value;

iii) less than the concentration used for the MDL study and the RL; and

iv) less than or equal to the RDL if applicable.

Table 10 shows an example of a practical range of detection for GenXusing 75 μL injection volumes.

TABLE 10 Example of a Practical Range of Detection for GenX. RL AnalyteName MDL RDL (MRL) MCL Trade Name IUPAC Name [μg/L] [μg/L] [μg/L] [μg/L]GenX 2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3- 0.0022 NA 0.010 50.0heptafluoropropoxy) propanoic acid

RDL (Required Detection Limit) detection limits are established byregulatory authority for certain analytes. The laboratory MDL valuesmust be equal to or lower than the RDL.

RL (Reporting Limits) or MRL (Minimum Reporting Level) is the practicaland routinely achievable values of analyte concentration. ReportingLimits determination is based on MDL values (see below). As used herein,there is no significant difference between the terms or abbreviations“reporting limits” or “RL,” and the terms or abbreviations “minimumreporting level” or “MRL”. Both terms and their abbreviations indicatepractical and routinely achievable values of analyte concentration.

MCL (Maximum Contamination Limit) represents the highest concentrationof analyte that is allowed in drinking water. The MCL values areestablished by regulatory authority. In the absence of an establishedMCL, 50 μg/L is typically a default limit for PFAS.

In this example with the method and system described supra, GenX isdetectable in water when the concentration of GenX is at least 0.0022μg/L using a 75 μL injection volume. This means that GenX is detectablein water if GenX has a concentration of 2.2 ppt or better with a 75 μLinjection volume. In terms of GenX amount, at least 1.7×10⁻⁷ μg of GenXin a single injection is detectable for any injection involving, forexample, an injection volume between approximately 1 μL and 100 μL.

In this example with the method and system described supra, a GenXconcentration in water is determinable when the concentration of GenX isat least 0.010 μg/L using a 75 μL injection volume. This means that aconcentration of GenX of 10 ppt in water is determinable with a 75 μLinjection volume. In terms of GenX amount, at least 7.5×10⁻⁷ g of GenXin a single injection is quantifiable for any injection involving, forexample, an injection volume between approximately 1 μL and 100 μL.

Determination of RL/MRL

In an embodiment, the RL (or MRL) is established for each method/analyteusing its calculated MDL value. In this embodiment, the RL/MRL is set ata value of 1 to 5 times the MDL value and then this set RL/MRL value isconfirmed as described below. In another embodiment, the RL/MRL may beset at a Required Detection Limit (RDL), which is a limit that is set bya regulatory authority. In this case, the RL/MRL based on the RDL isstill confirmed in the same way as an RL/MRL based on the calculated MDLvalue (as described below).

The RL/MRL is confirmed by processing and analyzing seven replicates oflaboratory fortified blanks (LFB) that are fortified with analyte at orbelow the set RL/MRL concentration. The LFB also include allmethod-specified dechlorination agents (e.g., ammonium chloride) andpreservatives, which are included in typical sample preparation.

First, the results of the analytical run are used to determine the meanconcentrations of the LFB and their standard deviations.

Second, the Half Range for the prediction interval of results (HR_(PIR))are calculated using the following equation:

HR _(PIR)=3.963×S

Where:

-   -   S=standard deviation of the seven LFB concentration        measurements; and 3.963 is the factor specific to seven        replicates.

An upper and lower limit of the Prediction Interval of Result(PIR=Mean±HR_(PIR)) provides confirmation of the set RL/MRL if it meetstwo criteria: The Upper PIR Limit (PIR_(UL)) must be ≤150% recovery andThe Lower PIR Limit (PIR_(LL)) must be ≥50% recovery for the RL/MRL tobe confirmed. The calculations for PIR_(UL) and PIR_(LL) are as follows:

${PIR}_{UL} = {{\frac{{Mean} + {HP}_{PIR}}{{Fortified}\mspace{14mu} {Concentration}} \times 100} \leq {150\%}}$${PIR}_{LL} = {{\frac{{Mean} - {HP}_{PIR}}{{Fortified}\mspace{14mu} {Concentration}} \times 100} \geq {50\%}}$

In various embodiments using a 75 μL injection volume, the LC/MS/MSmethod and system as described herein is used to detect GenX in aqueoussamples with concentrations of GenX as low as approximately 0.0022 μg/L(i.e., the calculated value of the MDL). In various embodiments using a75 μL injection volume, the LC/MS/MS method and system as describedherein is used to detect GenX in aqueous samples with concentrations ofGenX as low as approximately 2.2 ppt. In various embodiments, theLC/MS/MS method and system as described herein is used to detect GenX inan injection volume between approximately 1 μL and 100 μL that containsat least approximately 1.7×10⁻⁷ μg of GenX. In particular, the LC/MS/MSmethod and system set with the parameters shown in Table 4 on an AGILENT6490 or AGILENT 6495 mass spectrometer are shown to detect levels ofGenX in aqueous samples with concentrations of GenX as low asapproximately 0.0022 μg/L.

In various embodiments using a 75 μL injection volume, the LC/MS/MSmethod and system as described herein is used to determine concentrationlevels of GenX in aqueous samples with concentrations of GenX as low asapproximately 0.010 μg/L (i.e., the value of the RL or MRL). In variousembodiments using a 75 μL injection volume, the LC/MS/MS method andsystem as described herein is used to determine concentration levels ofGenX in aqueous samples with concentrations of GenX as low asapproximately 10 ppt. In various embodiments, the LC/MS/MS method andsystem as described herein is used to determine an amount of GenX in aninjection volume between approximately 1 μL and 100 μL that contains atleast approximately 1.7×10⁻⁷ μg of GenX. In particular, a LC/MS/MSmethod and system set with the parameters shown in Table 4 on an AGILENT6490 or AGILENT 6495 mass spectrometer are shown to determineconcentration levels of GenX in aqueous samples with concentrations ofGenX as low as approximately 0.010 μg/L.

In various embodiments, an LC/MS/MS method and system as describedherein is used to determine concentration levels of GenX in aqueoussamples with concentrations of GenX as low as approximately 0.010 μg/Lwhen the ESI gas temperature setting is approximately 120° C. toapproximately 160° C. on an AGILENT 6490 or AGILENT 6495 massspectrometer.

In various embodiments, an LC/MS/MS method and system as describedherein is used to determine concentration levels of GenX in aqueoussamples with concentrations of GenX as low as approximately 0.010 μg/Lwhen the ESI sheath gas heater setting is approximately 150° C. toapproximately 275° C. on an AGILENT 6490 or AGILENT 6495 massspectrometer.

In various embodiments, an LC/MS/MS method and system as describedherein is used to determine concentration levels of GenX in aqueoussamples with concentrations of GenX as low as approximately 0.010 μg/Lwhen the ESI sheath gas flow is approximately 6 L/min to approximately11 L/min on an AGILENT 6490 or AGILENT 6495 mass spectrometer.

In various embodiments as described above using a 75 μL injectionvolume, the LC/MS/MS method and system as described herein is used todetermine concentration levels of GenX in aqueous samples withconcentrations of GenX as low as approximately 0.010 μg/L and as high asapproximately 1.0 μg/L. In various embodiments as described above usinga 75 μL injection volume, the LC/MS/MS method and system as describedherein is used to determine concentration levels of GenX in aqueoussamples with concentrations of GenX as low as approximately 10 ppt andas high as approximately 1000 ppt. In various embodiments as describedabove, the LC/MS/MS method and system as described herein is used todetermine amounts of GenX in an injection volume between approximately 1μL and approximately 100 μL that contains approximately 1.7×10⁻⁷ μg to1.7×10⁻⁵ of GenX. It will be readily apparent by a person of skill inthe art that samples which are too concentrated for accurateconcentration determination using methods and systems described herein,are diluted so that the diluted concentrations are accurately measured.The original sample concentrations are then calculated based well-knownequations for doing so, e.g. M₁V₁=M₂V₂.

In step 145, samples from various sources of water are collected andanalyzed using the above described method. In an example, samples arecollected in 250 mL polypropylene bottles. In another example, the 250mL bottles are pre-charged with approximately 50 mg of ammoniumchloride.

In an example for the analysis of GenX in tap water, the water tap isallowed to run freely until the water temperature has stabilized, andthe flow is reduced to permit bottle filling without splashing. Thebottle is filled to the neck, taking care not to flush out the ammoniumchloride, if present. The bottle is then capped and agitated to dissolvethe ammonium chloride, if present, and placed in a cooler with frozengel packs.

In some embodiments, the samples received at the laboratory on thecollection day are transported in coolers with frozen gel packs andtheir temperature is maintained between 1° C. and 10° C. for the first48 hours.

In other embodiments, the samples that will not be received at thelaboratory on the day of collection are maintained at a temperaturerange between approximately 1° C. to 6° C. until analysis is initiatedat a receiving laboratory.

In some embodiments, a maximum holding time from collection to analysisis 14 days.

In various embodiments, samples are prepared for analysis by removingfrom refrigeration and allowing the samples to equilibrate to ambienttemperature. In some embodiments, the samples are checked fordechlorination efficiency by testing with free chlorine strips to ensurethat the free chlorine level is <0.1 mg/L. Samples, standards, and QCsare next loaded into 2 mL autosampler vials. In some embodiments, thesamples and QCs are spiked with 10 μL of internal standard.

The samples are then analyzed by injection alongside the standards andQCs into an LC/MS/MS with ESI using the conditions described supra.

In an example, after the initial calibration is confirmed valid with theQCS and CCV, analyzing field and QC samples is typically begun at thefrequency outlined in Table 11 below. The instrument's MASS HUNTERsoftware is used in the calibration procedure.

TABLE 11 Example of Confirmation of Initial Calibration for GenX.Analysis Int. # Sample Name QCs, ICSs, CCVs Acceptance Criteria Std. QCand Instrument Calibration Frequency 1 ICS 1 1. Instrument Calibrationis updated and recalculated Internal Standard Analyzed with evelyanalytical run. 2 ICS 2 against the newly generated calibmtion curve.Response 3 ICS 3 2. Each analyte in each calibration point, except forthe Relative Percent 4 ICS 4 concentrations ≤ RL, must calculate to be±30% of the Deviation (ISRPD) 5 ICS 5 true value. must be ±50%. 6 ICS 63. Each analyte in calibration points at concentrations ≤ 7 ICS 7 RLmust calculate to be ±50% of the true value. 8 QCS 1. Recovery fortarget analytes must be ±30% of the Analyzed every time an instrumenttrue value. calibmtion is run at the beginning of an analytical run. 9CCV LL 1. Recovery for target analytes must be ±50% of the Analyzed atthe beginning of an analytical true value. batch. 10 MB 1. Must be freefrom contamination that could prevent Analyzed with each batch of up to20 the determination of any target analyte. samples processed as a groupwithin a 2. Concentration of target analytes must be ≤⅓ RL. work shift.11 Sample 1 12 LFM LFM/D: Recovery for target analytes should be ±40% ofAnalyzed with each batch of up to 20 13 LFMD the true value; precisionas RPD should be ≤30%. samples processed as a group within a work shift.14 Sample 2 15 Sample 3 16 Sample 4 17 Sample 5 18 Sample 6 19 Sample 720 Sample 8 21 Sample 9 22 Sample 10 23 CCV ML 1. Recovery for targetanalytes must be ±30% of the Analyzed with each analytical batch of uptrue value to 20 samples after the first 10 samples. 24 Sample 11 25Sample 12 26 Sample 13 27 Sample 14 28 Sample 15 29 Sample 16 30 Sample17 31 Sample 18 32 Sample 19 31 Sample 20 32 CCV HL 1. Recovery fortarget analytes must be ±30% of the Analyzed with each analytical batchof up true value to 20 samples after the second 10 samples. RL =reporting limits.

In this example, MASS HUNTER analytical software uses peak areas and theinternal standard technique to calculate concentrations of the methodanalytes. Data may be fit with either a linear or quadratic regressionwith weighting if necessary.

In this example, the percent recovery calculation for CCV, LFB, QCS andLFM is performed using the following formula:

$P = {\frac{A - B}{T} \times 100\%}$

Where:

-   -   P=percent recovery;    -   A=measured concentration of analyte after spiking;    -   B=measured background concentration of analyte; and    -   T=true concentration of the spike.

In this example, relative percent difference for the fortified matrixduplicate is calculated using the following formula:

${RPD} = {\frac{{{LFM} - {LFMD}}}{\frac{{LFM} + {LFMD}}{2}} \times 100\%}$

Where:

-   -   RPD=relative percent difference;    -   LFM=measured concentration of analyte in the fortified sample;        and    -   LFMD=measured concentration of analyte in the fortified sample        duplicate.

In this example, Internal Standard Response Relative Percent Deviation(ISRPD) is calculated as follows:

${ISRPD} = {\frac{\begin{matrix}{{{IS}\mspace{14mu} {Response}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {Sample}} -} \\{{Average}\mspace{14mu} {IS}\mspace{14mu} {Response}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {Initial}\mspace{14mu} {Calibration}}\end{matrix}}{{Average}\mspace{14mu} {IS}\mspace{14mu} {Response}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {Initial}\mspace{14mu} {Calibration}} \times 100}$

The LC/MS/MS is configured to determine a concentration of GenX asdescribed supra. In particular, the various ESI conditions describedabove in the detailed embodiments are utilized.

The LC/MS/MS outputs data that allows the determination of aconcentration of GenX within the unconcentrated sample, wherein theconcentration of GenX within the unconcentrated sample has a minimumreporting level of approximately 0.010 μg/L.

FIG. 2 depicts a block diagram of components 200 of an LC/MS/MS systemused to determine levels of GenX in samples in accordance with anexemplary embodiment of the present invention. It should be appreciatedthat FIG. 2 provides only an illustration of one implementation and doesnot imply any limitations with regard to other systems in whichembodiments of the present invention may be implemented. Othermodifications to the depicted system may be made without departing fromthe scope of the present invention.

LC/MS/MS system 200 includes injector 210, LC column 215, ESI ionizercomponent 220, triple quadrupole mass spectrometer (TQMS) component 225,ion detector 230, and mass spectrum read-out software 235.

TQMS 225 includes two quadrupole mass analyzers in series (225Q1 and225Q3) with a non-mass-resolving quadrupole (225Q2) between them to actas a cell for collision-induced dissociation. All three quadrupole massanalyzers consist of four cylindrical rods (for reasons of simplicitythey are schematically represented by the labeled parallel bars in FIG.2.). The four cylindrical bars are set parallel to each other. For 225Q1and 225Q3, each opposing rod pair is connected together electrically anda radio frequency (RF) voltage with a DC offset voltage is appliedbetween one pair of rods and the other. Ions travel down the quadrupolebetween the rods. Only ions of a certain mass-to-charge ratio will reachdetector 230 for a given ratio of voltages. Other ions have unstabletrajectories and will collide with the rods. This permits selection ofan ion with a particular m/z or allows the operator to scan for a rangeof m/z-values by continuously varying the applied voltage. Quadrapole225Q2 is an RF-only quadrupole (non-mass filtering) for collisioninduced dissociation of selected parent ion(s) from 225Q1. Subsequentfragments are passed through to 225Q3 where they may be filtered orfully scanned.

In an embodiment, an aliquot of GenX sample 205 is injected intoinjector 210 and the injection liquid 201 is separated from othernon-GenX analytes by LC column 215 using, for example, the column,conditions, and gradient example shown and described for Table 5.

After eluting through LC column 215, the GenX-containing eluent 202 issubjected to ESI 220. As stated supra and reiterated here, ESI is atechnique used in mass spectrometry to produce ions using anelectrospray in which a high voltage is applied to a liquid to create anaerosol that is ionized. Conditions for ionization of GenX using the ESItechniques as embodied by ESI 220 have been detailed and described suprain embodiments of the present invention.

After ionization of the GenX-containing eluent by ESI 220, the ion(s)203 are passed through the first quadrupole mass analyzer, 225Q1, whichserves as a filter for selecting desired GenX ions 204. The secondquadrupole mass analyzer, 225Q2, allows for collision of selected ions204 to produce one or more children ions 206 that then pass through thethird quadrupole mass analyzer, 225Q3. Quadrupole mass analyzer 225Q3provides a scan of the entire m/z range of the product ion(s) 206,providing output 207 for fragments 206. Quantification of selected ion204 can then be deduced from the ion fragmentation output 207 receivedby ion detector 230 and processed by mass spectrum read-out software235.

It should be appreciated that all combinations of the foregoingembodiments and additional embodiments discussed in greater detailherein (provided such embodiments are not mutually inconsistent) arecontemplated as being part of the inventive subject matter disclosedherein. In particular, all combinations of claimed subject matterappearing at the end of this disclosure are contemplated as being partof the inventive subject matter disclosed herein.

Although the invention has been described by reference to specificexamples, it should be understood that numerous changes may be madewithin the spirit and scope of the inventive concepts described.Accordingly, it is intended that the disclosure not be limited to thedescribed examples, but that it have the full scope defined by thelanguage of the following claims.

1. A method for detecting GenX in a sample comprising: injecting asample into an LC/MS/MS system that is configured to detect GenX withinthe sample, wherein the LC/MS/MS includes ESI; subjecting the sample toESI conditions that include a sheath gas flow of approximately 6 L/minto approximately 11 L/min; and detecting GenX within the sample.
 2. Themethod of claim 1 further comprising: determining a concentration ofGenX within the sample that is at least approximately 0.010 μg/L.
 3. Themethod of claim 1, wherein the ESI conditions further comprise a sheathgas heater setting of approximately 150° C. to approximately 275° C. 4.The method of claim 1, wherein the ESI conditions further comprise aprobe gas temperature of approximately 120° C. to approximately 160° C.5. The method of claim 1, wherein a detectable concentration of GenXwithin the sample is at least approximately 0.0022 μg/L.
 6. The methodof claim 1, wherein a detectable amount of GenX in a single injection ofthe sample is at least approximately 1.7×10⁻⁷ g.
 7. A method fordetecting GenX in a sample comprising: injecting a sample into anLC/MS/MS system that is configured to detect GenX within the sample,wherein the LC/MS/MS includes ESI; subjecting the sample to ESIconditions that include a sheath gas heater setting of approximately150° C. to approximately 275° C.; and detecting GenX within the sample.8. The method of claim 7 further comprising: determining a concentrationof GenX within the sample that is between approximately 0.010 μg/L toapproximately 1.0 μg/L.
 9. The method of claim 7 further comprising:determining a concentration of GenX within the sample that is at leastapproximately 0.010 μg/L.
 10. The method of claim 7, wherein the ESIconditions further comprise a sheath gas flow of approximately 6 L/minto approximately 11 L/min.
 11. The method of claim 7, wherein the ESIconditions further comprise a probe gas temperature of approximately120° C. to approximately 160° C.
 12. The method of claim 7, wherein adetectable concentration of GenX within the sample is at leastapproximately 0.0022 μg/L.
 13. The method of claim 7, wherein adetectable amount of GenX in a single injection of the sample is atleast approximately 1.7×10⁻⁷ g.
 14. A GenX detection system comprising:an LC/MS/MS system operable utilizing ESI and configured to: receive aninjection of a sample containing GenX; subject the sample to ESIconditions that include a sheath gas flow of approximately 6 L/min toapproximately 11 L/min; and detect GenX within the sample.
 15. Thesystem of claim 14, wherein the LC/MS/MS system is configured todetermine a concentration of GenX within the sample that is at leastapproximately 0.010 μg/L.
 16. The system of claim 14, wherein the ESIconditions further comprise a sheath gas heater setting of approximately150° C. to approximately 275° C.
 17. The system of claim 14, wherein theESI conditions further comprise a probe gas temperature of approximately120° C. to approximately 160° C.
 18. The system of claim 14, wherein adetectable GenX concentration in the sample is at least approximately0.0022 μg/L.
 19. The system of claim 14, wherein a detectable amount ofGenX in a single received injection of sample is at least approximately1.7×10⁻⁷ g.
 20. A method for facilitating the detection of GenX in asample comprising: obtaining a sample containing GenX; receiving datarepresentative of test results of an analysis detecting GenX within atleast a portion of the sample, wherein the analysis comprised thefollowing steps a) and b): a) injecting a volume of the sample into anLC/MS/MS system with ESI that is configured to detect GenX; and b)subjecting the injected volume of the sample to ESI conditions thatinclude a sheath gas flow of approximately 6 L/min to approximately 11L/min.